Interpretive Summary: Eleusine indica (goosegrass) represents one of the most problematic weeds worldwide. The second weed biotype resistant to the herbicide Roundup appeared in Malaysia, and it was a biotype of Eleusine indica. In our study we examined the target enzyme EPSP synthase’s sensitivity to glyphosate, EPSP synthase gene expression, and genomic organization of EPSP synthase gene variants in sensitive and resistant plants. Our results indicate no significant differences in EPSPS gene expression or gene copy number. Reduced sensitivity of the EPSPS enzyme to the herbicide glyphosate was however observed in resistant plants. Comparative sequence analysis of EPSP synthase variants in resistant and sensitive plants identified two amino acid changes in the resistant EPSPS enzyme. Further mutagenesis experiments showed that only one of the mutations (a proline to serine substitution) conferred resistance to the herbicide. This report represents the first instance where the likely molecular basis for resistance to glyphosate has been determined for any weed species.

Technical Abstract:
The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD50 value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC50(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.